Nine cyclic nucleotide phosphodiesterase gene families (PDE1-9) have been identified. PDE3 isoforms are characterized by their high affinity for cAMP and cGMP, their specific inhibition by certain drugs that increase myocardial contractility, relax airway and vascular smooth muscle, inhibit platelet aggregation and stimulate insulin secretion, and their rapid activation in response to insulin, IGF-1, IL-4, and agents that increase cAMP. Two PDE3 families, PDE3A and PDE3B, have been identified. A second human (H) PDE3B isoform, HPDE3B2, was cloned from a human Jurkat cell cDNA library. The open reading frame of HPDE3B2 is virtually identical to that of HPDE3B1; these isoforms differ in the sequences of their 3' UTRs and may reflect the presence of 2 mRNA species observed in Northern Blots of multiple human tissue mRNAs with HPDE3B probes. A possible splice variant of PDE3A was cloned from a porcine aorta smooth muscle cDNA library; its predicted molecular weight was similar to that of a PDE3 purified from bovine aorta. Structure/function studies with wild type and N- and C-terminal truncated recombinants of PDE3A and B indicated that 1) the PDE3 catalytic core includes the C-terminal domain [approx. 270 aa (amino acids)]conserved among all mammalian PDEs plus some additional upstream and downstream sequences; 2) the N-terminal half of PDE3 is not required for catalytic activity or sensitivity to specific inhibitors. HPDE3Adel190(in which the first 190 aa were deleted) exhibited an IC50 value 4 fold lower than that for wild type PDE3A, suggesting that the N-terminal portion of PDE3A can regulate responsiveness to at least this specific PDE3 inhibitor; and 3) the N-terminal hydrophobic region of PDE3 which contains 5-6 predicted transmembrane helices is important in association of PDE3 with, or targeting to, intracellular membranes. Immunofluorescent microscopy using an antibody raised against the N-terminal sequence of PDE3B indicated that in cultured 3T3-L1 adipocytes, PDE3B exhibited strong reticular staining which co-localized with the endoplasmic reticulum (ER) marker protein BIP. These studies are consistent with those reported last year with N-terminal truncated recombinants expressed in both COS cells and Sf9 insect cells which suggested that the hydrophobic region (and transmembrane segments) is important in association of PDE3 with the ER. Gel filtration studies also indicated that whereas the N-terminal hydrophobic region promoted formation of large PDE3 aggregates, it was not necessary for oligomerization since recombinants in which the first 511 aa of HPDE3A were deleted eluted as a dimer during chromatography on Ultrogel AcA54. To learn more of PDE3 function we are attempting to disrupt PDE3A and B genes by homologous recombination in mice. Heterozygous chimeric mice in which the PDE3B gene has been successfully disrupted have been generated and we are now attempting to produce homozygous mice. Promoter activity in the approx. 5 kb of the 5' flanking region of the mouse PDE3B gene has been identified with a strong upstream promoter separated from a much weaker downstream promoter by at least 2-3 kb of an inhibitory or suppressor region.